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ACTH peptides stimulate motor nerve sprouting in development
EXPERIMENTALNEUROLOGY
lw,531-541(1988)
ACTH Peptides Stimulate Motor Nerve Sprouting in Development RUTH E. FRISCHER AND FLEUR L. STRAND’ Center for Neuroscience, New York University, Washington Square, New York, New York 10003 Received September 8, I987 Maturational changes at the neuromuscular junction (nmj) of rat neonates were studied using scanning electron microscopy and light microscopy that permitted quantification of muscle fiber diameter, length of nerve terminal branching, end-plate area, and perimeter. Administration of ACTH 4- 10 ( 10 &kg S.C.daily from day of birth) stimulated nerve terminal branching, an effect most evident in 14&y-old pups. The trisubstituted derivative of ACTH 4-9 (Org 2766) when administered at 0.01 pg/ kg/daily, had a more potent effect, increasing end-plate perimeter and nerve terminal branching on the first postnatal week and markedly increasing only nerve terminal branching at 14 days of age. This is a dose-responsive action since 10 &kg/daily severely inhibits nerve sprouting. By 2 1 days, there were no differences between peptide- and saline-treated neonates. Peptide-induced sprouting was elicited only in the first 2 weeks of postnatal life. This time course corresponds with the critical period for nmj maturation and ceases when polyneuronal innervation of muscle fibers also terminates. It is suggested that ACTH peptides may exert a physiological role on nerve sprouting during development. 0 1988 Academic PI=. 1~.
INTRODUCTION Nerve sprouting occurs as a physiological process during normal development; it occurs as a restorative process following nerve crush or section. In the newborn rat each muscle fiber is polyneuronally innervated; i.e., each muscle fiber is contacted by several motor nerves. Typically during maturaAbbreviations: EDL-extensor digitorum longus, LM-light microscopy, nmj-neuromuscular junction, SEM-scanning electron microscopy. ’ This study was supported by The Council for Tobacco Research and by Grant BRSG RR07062 awarded by the Biomedical Research Support Grant Program of Research Resources, National Institutes of Health. We also thank Organon for ACTH 4-10 (ORG 0163) and ORG 2766. 531 0014-4886188 $3.00 Copyright 0 1988 by Academic Press, Inc. All rights of repmdwtion in any form reserved.
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tion, the adult form of mono-neuronal innervation occurs between 2 and 3 weeks after birth (5, 18). In both intact adult nerve (28) and regenerating nerve (2,29,3 1) the presence of ACTH or ACTH peptide fragments stimulates the formation of nerve sprouts. While the causes of sprout formation during the process of regeneration appear to be better understood, the underlying mechanisms involved during development remain elusive. Both our electrophysiological studies and our studies on the development of motor behavior indicate that maturation of the neuromuscular system is accelerated by ACTH peptides ( 19,20,22). Using light and scanning electron microscopy (SEM) we have also shown that ACTH 4-10 and the ACTH 49 analog, Org 2766 (approximately 1000X more potent than ACTH 4-lo), have distinct neurotrophic effects, accelerating the morphological maturation of the developing rat neuromuscular junction (nmj) (10). In this study we asked whether this well-documented early maturation might be achieved through ACTH peptide effects on some phase of development such as axonal sprouting. We compared the effects of the two peptide fragments ACTH 4-10 and Org 2766, on structural parameters of the neuromuscular junction, including nerve terminal branching at the end-plate region, in neonatal rats 7, 14, and 2 1 days of age. METHODS
Animal Groups and Peptide Administration. Sprague-Dawley rats (dam plus pups) were maintained on a 12-h light/dark cycle and at a room temperature of 22°C. Each dam was housed separately and supplied with rat chow and water ad lib. On the day of birth (Day 1) each litter was culled to eight pups with approximately equal numbers of males and females. The pups were assigned randomly to litters and treated according to one of the following four protocols: 1. 0.9% saline 2. ACTH 4-10 (Org 0163), 10 pg/kg 3. Org 2766, the trisubstituted analog of ACTH 4-9,O.Ol &kg 4. Org 2766, 10 pg/kg was administered only in the series of experiments devoted to a SEM study of nerve terminal branching. As this dosage was found to be inhibitory, the lower dosage of 0.0 1 &kg, comparable to 10 &kg of ACTH 4- 10, was used for all other experiments. All injections were administered S.C. daily from Day 1. The animals were sacrificed at 7, 14, or 2 1 days of age. A series of untreated adult EDL muscles were processed for SEM for comparison purposes.
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Surgical Procedure. Pups were anesthetized with 40 mg/kg sodium pentabarbital i.p. before surgery. The animal was placed ventral side down on a dissecting table, a skin incision was made on the hind limb from the lateral epicondyle of the femur to the lateral malleolus in the ankle. The extensor digitorum longus (EDL) muscle was then exposed from the surrounding connective tissue. One EDL muscle was removed for the SEM studies, the contralateral muscle being used for light microscopy (LM). SpecimenPreparation for SEM. The muscle was fixed in situ and prepared for SEM using modifications of both Desaki and Uehara (8) and Fahim et al. (9). Endplates were localized with a cholinesterase stain, dissected free, postfixed in 2% unbuffered osmium tetroxide, and rinsed in distilled water. The extraneous cutaneous tissue was removed with 8 N HCl treatment in 60°C until the tissue separated into individual muscle fibers. The fibers were collected with a 5-ml glass syringe and allowed to flow onto an 0.8-pm poresize Nucleopore filter. The specimens were then washed with distilled water, dehydrated through a graded series of ethanol, critical-point dried, coated with carbon and gold palladium, and examined in an AMR 1000 scanning electron microscope at 20 kV. Specimen Preparation for LM. Intramuscular nerves and end plates were visualized by the combined silver-cholinesterase method of Pecot-Dechavassine et al. (17). The EDL muscle was fixed in situ by the application of 1% neutral formalin in normal Ringer’s solution. After excision, the EDL muscle was placed in fresh 1% neutral formalin for 3-4 min. The end plates were localized with a cholinesterase stain. The whole muscle was fixed in 5% neutral formalin, placed in 10% Triton X- 100, and postfixed in 80% ethanol. After impregnation with 0.5% Protargol the silver stain was reduced by the use of a modified Bodian developer. The whole muscle was rinsed in distilled water, finely teased, and mounted on slides. Quantitative Morphometric Analyses of LM Preparations. LM preparations were observed using an Olympus BH 100X oil immersion objective. A low-light-sensitive video camera mounted on top of the microscope projected the slide image onto a monitor. A Bioquant image digitizer with a manually operated cursor was used to determine measurements. Parameters measured included muscle fiber diameter, total length of nerve terminal branching, and cholinesterase-positive end-plate area and perimeter. Eight nmjs per neonate were measured. Each group consisted of five neonates for a total of 40 nmjs per group. An example of an outline graphics of digitized nmjs is demonstrated in Fig. 1. Data and outline graphics of all end-plate measurements were automatically fed into an Apple IIe computer. The Student t test was used for statistical evaluation. Data are expressed as means and standard error of the mean.
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FIG. 1. An example of a computer graphic outline of neuromuscular junctions after obtaining morphometric parameters. The numbers on the outline correspond to one of eight neuromuscular junctions measured.
RESULTS Scanning Electron Microscopy. During development there is a progressive change in the pattern of innervation of the muscle. The polyneuronal innervation of the immature skeletal muscle is shown in Fig. 2A. For comparison, the innervation of each muscle fiber by a single nerve, the characteristic pattern of the adult motor unit is shown in Fig. 2B. Figure 2C indicates the complexity of nerve terminal attachments at the adult nmj. When neonatal animals are treated with ACTH 4-10 (10 &kg) or Org 2766 (0.01 pg/kg) during the first 2 weeks of life, arborization of the nerve terminals entering the EDL is markedly increased (Fig. 3C-E) as compared to saline-treated controls (Fig. 3A). The inhibitory effect of the higher dosage of ORG 2766 (10 pg/kg) on nerve branching is clearly seen in Fig. 3B. Quantification of the three dimensional views of the developing end plates is difficult: peptide treatment appears to increase nerve terminal branching, a subjective interpretation that is borne out by the morphometric analysis of the LM preparations. Morphometric Analysis of LM Preparations. Table 1 shows that as the saline-treated neonate matures from 7 to 14 to 2 1 days of age, there are clear increases in muscle fiber diameter, end-plate area and perimeter, and nerve terminal branching. Treatment with ACTH 4- 10 ( 10 pg/kg) results in a diminution of muscle fiber diameter (P < 0.01) when measurements are made at 7 days of age. End-plate area and nerve terminal branching are similarly reduced (P < 0.05) at this time in development. No effect on end-plate perimeter is evident. However, Org 2766 at this stage in development increases
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FIG. 2. A-several nerves converging at a neuromuscular junction site on EDL muscle fiber of 7-day-old rat. 2440X. B-a motor unit of the peroneal nerve is seen branching and innervating several EDL muscle fibers. 220X. C-an adult rat end plate showing the complexity of nerve terminal attachment. 3050X.
both end-plate perimeter and nerve terminal branching. When peptide treatment is continued to Day 14, it is clear that both ACTH peptides stimulate nerve terminal branching, with Org 2766 being considerably more effective
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FIG. 3. Scanning electron micrographs of end-plate regions from the EDL muscles of 15 day-old rats. A-saline-treated neonate. The nerve can be seen branching into narrower nerve terminals along the muscle fiber. 188 1X. B-ORG 2766-treated (10 &kg) neonate. The nerve terminal shows a marked reduction in branching. 1986X. C-ACTH 4-lo-treated (10 pplkg) neonate showing extensive nerve terminal branching. 1100X. D-ORG 2766~treated (0.01 ag/ kg) neonate with arborization of nerve terminals. 2750X. E-higher magnification of ORG 2766-treated (0.01 &kg) neonate showing considerable nerve terminal branching at the end plate. 5775X.
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TABLE I Endplate Measurements from EDL Muscles of Rats Treated with ACTH/MSH 4- 10 (10 &kg), ORG 2766 (0.01 &kg), or Saline Daily S.C.from Day of Birth Age 7 Days Saline 4-10 2766 14 Days Saline 4-10 2766 2 1 Days Saline 4-10 2766
Muscle fiber diameter
End-plate area
End-plate perimeter
(rm)
(wd
(0)
Nerve terminal branching (gm)
32.1 +- 0.7 29.4 f 0.5** 32.9 f 0.9
1010.8 + 42.1 907.2 + 27.7* 1084.0 f 40.6
126.1 _t 2.1 122.3 k 2.2 137.7 f 2.5’*
108.3 -e 3.9 94.6 + 3.8+ 122.2 f 4.1*
38.2 + 0.9 38.2 + 0.9 38.5 f 0.9
1556.8 + 82.9 1631.9 f 63.6 1792.9 f 104.8
160.5 k 4.0 159.0 + 2.9 166.9 + 4.8
146.4* 6.7 172.4 + 8.0** 192.7 + 7.7***
55.1 * 1.2 53.9 f 1.4 52.3 + 1.1
2721.8 + 108.6 2686.0 k 101.8 2942.6 + 101.5
209.3 + 4.2 211.1 t4.1 220.4 + 4.2
296.6 + 8.3 273.6 i 10.1 294.6 f 8.7
Note. Values are means f SE. n = 40. * P < 0.05. **p
(P < 0.001) than ACTH 4-10 (P < 0.05) in this regard (Table 1). However, at this age there is little difference between the saline-treated controls and those animals administered either ACTH peptide when muscle fiber diameter, end-plate area, and perimeter are analyzed. By 2 1 days animals administered either ACTH peptide (when muscle fiber diameter, end-plate area, and perimeter are analyzed) do not differ from the saline controls in any of these morphological parameters. DISCUSSION These results confirm our earlier observations that ACTH 4- 10 is primarily neurotrophic in action (13,27). We show here that of all the parameters investigated, only nerve terminal branching at 14 days of age is effectively stimulated by this peptide. Muscle fiber diameter and end-plate area are actually reduced at 7 days of age and are subsequently unresponsive to ACTH 4- 10. The ACTH 4-9 analog, Org 2766, on the other hand, does not evoke these early presumably deleterious effects on muscle fiber diameter and endplate area, increasing both end-plate perimeter and nerve terminal branching at 7 days of age and stimulating nerve terminal branching still more effectively at 2 weeks of age. This action is dose-responsive, with 0.01 &kg being
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stimulatory and 10 pg/kg markedly inhibitory, a result often incurred with peptide administration. Electrophysiological studies clearly demonstrate that Org 2766 improves both the quality and rapidity of muscle reinnervation (24). During both development and regeneration, the presence of ACTH or ACTH peptide fragments stimulates the formation of nerve sprouts. In the developing neuromuscular system, this is seen as an increase in peripheral axonal arborization at the end-plate region (1). The present studies on the developing nmj describe an age-related increase in nerve terminal branching both prior to entering the immature muscle (Fig. 3) and within the end plate (Table l), correlating to the period of polyneuronal innervation. Therefore, the process of arborization is enhanced by peptide treatment during the first 2 weeks of postnatal life, with Org 2766 being a more potent stimulus for nerve branching than ACTH 4-10. Stimulatory effects of ACTH peptides on nerve outgrowth following nerve crush have been demonstrated by Gispen and his colleagues (3,7,3 1). These investigators also reported that of the melanocortin family of peptides, a-MSH (ACTH 1- 13) and its analogs have more potent neurotrophic properties than the corticotrophic moiety ACTH 4- 10 or its fragments. None of these peptides have any significant adrenocortical-stimulating activity ( 15). One of several possible modes of action to explain the acceleration of nerve sprouting induced by ACTH peptides in both development and regeneration involves a suggestion put forward by Gispen (12): namely, that following binding by the peptide to nerve-specific sites, calcium-dependent phosphorylation of specific synaptic plasma membrane proteins is inhibited. One of these proteins is B-50, which appears to be identical to GAP-43 and protein F 1 ( 16) phosphoproteins also associated with synaptic membranes. B-50 immunoreactivity is considerably increased in the regenerating peripheral nerves and localized in their newly formed sprouts and in the neuromuscular junctions (32). Of interest to any hypothesis that attempts to correlate mechanisms involved in regeneration and development is that maximal B-50 synthesis corresponds approximately to periods of profuse axonal outgrowth and synaptogenesis during ontogeny. During the development of the mammalian central nervous system, B-50 synthesis peaks early in the second postnatal week (14,25). It is also during these crucial first 2 weeks of postnatal life that the infant rat neuromuscular system is responsive to ACTH peptide administration ( 1, 10, 11,20-22,26). Synthesis of this growth-associated protein rapidly declines after this time, as does susceptibility to peptide action. The problem with this concept is that B-50 phosphorylation has not been shown to be sensitive to the smaller ACTH fragments such as ACTH 4- 10; sensitivity in the regenerating nerve appears to be limited to wMSH (ACTH l-l 3) and ACTH l-24 (33). The possibility that the developing nervous system may
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respond to the smaller ACTH fragments remains to be investigated. Other possible mechanisms that may explain our results include the suggestion that ACTH may evoke axonal sprouting at critical developmental periods by influencing neuronal responsiveness to some local target-tissuereleased trophic substance (4). ACTH may act in concert with a trophic substance and thus synergistically evoke nerve terminal sprouting by generating a permissive environment for sprouting to occur. ACTH can act only briefly in this capacity as there is a very small window in time that is open in the course of nmj development wherein sprouting can occur. It has been clearly shown that in regenerating systems, ACTH peptides effectively accelerate motor and sensory recovery only if peptide administration is begun within 48 hr after nerve crush (6,24). As administration immediately following the lesion is still better, it would appear that the melanocortins act on early growth processes (3 1) and that sensitivity to these peptides is indeed timedependent. In this morphological study all peptide-induced changes are evident only during the first 2 weeks of postnatal life: peptide acceleration of nmj maturation brings these parameters to a level at 2 weeks of age which is reached by the saline-treated controls at only 3 weeks of age. This demonstrates once again, as in our behavioral and electrophysiological studies (1, 19,20-22,27, 30), that neuropeptides bring a developing, regenerating, or depressed system to an optimal physiological level but do not elevate it to superlevels. SUMMARY In this paper we report that ACTH 4-10 and ORG 2766 stimulate nerve terminal sprouting most significantly at 14 days of age in the rat. The time course of this effect correlates with the maturational processes involved in neuromuscular development and ceases during the termination of polyneuronal innervation. This suggests that the stimulation for motoneuron terminal sprouting can be facilitated and further promoted by the exogenous administration of ACTH peptides at critical developmental periods and indicates a physiological role for these neurotrophic peptides in nerve sprouting. REFERENCES 1. ACKER, G. R., R. E. FRISCHER,AND F. L. STRAND. 1984. ACfH peptide neuromodulation in the developing neuromuscular system as seen through three different perspectives. Ann. N. Y. Acad. Sci. 435: 370-375. 2. BJJLSMA, W. A., F. G. I. JENEKENS, P. S~HOTMAN, AND W. H. GISPEN. 1983. Stimulation by ACTH 4- 10 of nerve fiber regeneration following sciatic nerve crush. Muscle Nerve 6: 104-l 12. 3. BIJLSMA, W. A., P. SCHOTMAN, F. G. I. JENEKENS,W. H. GISPEN, AND D. DE WIED. 1983. The enhanced recovery of sensorimotor function in rats is related to the melanotropic moiety of ACTH/MSH neuropeptides. Eur. J. Pharmacol. 9: 231-236.
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4. BROWN, M. C., R. L. HOLLAND, AND W. H. HOPKINS. 198 I. Motor nerve sprouting. Annu. Rev. Neurosci. 4: 11-42. 5. BROWN, M. C., J. K. S. JANSEN, AND D. VAN ESSEN. 1976. Polyneural innervation of skeletal muscle in new born rats and its elimination during maturation. J. Physiol. 261: 387-422. 6. DEKKER, A. J. A. M., M. M. PRINCEN,H. DE NIJS, L. G. J. DE LEEDE, AND C. L. E. BROEKKAMP. 1987. Acceleration of recovery from sciatic nerve damage by the ACIH (4-9) analog Org 2766: Different routes of administration. Peptides 8: 1057-1060. 7. DE KONING, P., J. H. BRAKEE, AND W. H. GISPEN. Methods for producing a reproducible crush in the sciatic nerve of the rat and the rapid and precise testing of return of sensory function: Beneficial effectsof melanocortins. J. Neurol. Sci. 14: 237-246. 8. DESAKI, J., AND Y. UEHARA. 198 1. The overall morphology of neuromuscular junctions as revealed by scanning electron microscopy. J. Neurocytol. 10: 10 1- 110. 9. FAHIM, M. A., J. A. HOLLEY, AND N. ROBBINS. 1983. Scanning and light microscopic study of age changes at a neuromuscular junction in the mouse. J. Neurocytol. 12: 12-25. 10. FRISCHER,R. E., N. EL-KAWA, AND F. L. STRAND. 1985. ACTH peptides as organizers of neuronal patterns in development: Maturation of the rat neuromuscular junction as seen by scanning electron microscopy. Peptides 6(Suppl. 2): 13- 19. 11. FRISCHER,R. E., AND F. L. STRAND. 1988. Neural effects of ACTH peptide treatment on the developing rat neuromuscular junction. Ann. N. Y. Acad. Sci., in press. 12. GISPEN, W. H., C. J. VAN DONGEN, P. N. E. DEGRAAN, A. B. OFSTREICHER, AND H. ZWIERS. 1985. The role of phosphoprotein B-50 in phosphoinositide metabolism in brain synaptic plasma membranes. Pages 399-413 in J. E. BLEASDALE, G. HAWSER, AND J. EICHBERG,Eds., Inositol and Phosphoinositides. Humana, Clifton, NJ. 13. GONZALEZ, E. R., AND F. L. STRAND. 198 1. Neurotropic action of ACTH/MSH 4- 10 on neuromuscular function in hypophysectomized rats. Peptides 2(Suppl. 1): 107- 113. 14. JACOBSON,R. D., I. VIRAG, AND J. H. P. SKENE. 1986. A protein associated with axon growth, GAP-43, is widely distributed and developmentally regulated in rat CNS. J. Neurosci. 5: 1843-1855. 15. LANDE, S., D. DE WIED, AND A. WITHER. 1973. Unique pituitary peptides with behavioraffecting activity. Prog. Brain Res. 39: 42 l-427. 16. NELSON, R. B., AND A. ROUTTENBERG. 1985. Characterization of protein Fl(47 kDa, 4.5 pl): A kinase C substrate directly related to neural plasticity. Exp. Neurol. 89: 2 13-224. 17. PECOT-DECHAVASSINE, M., A. WERNIG, AND H. STOVER. 1979. A combined silver and cholinesterase method for studying exact relations between the pm- and the postsynaptic elements at the frog neuromuscular junction. Stain Technol. 54: 25-27. 18. REDFERN, P. A. 1970. Neuromuscular transmission in newborn rats. J. Physiol. 209: 701709. 19. ROSE, K. J., R. E. FRISCHER,J. A. KING, AND F. L. STRAND. Neonatal neuromuscular parameters vary in susceptibility to postnatal ACTH 4-10 administration. Peptides, in press. 20. ROSE, K. J. AND F. L. STRAND. 1987. Response of the developing neuromuscular system of the rat to nicotine and the neurotropic peptide fragment ACTH/MSH 4-10. Ann. N. Y. Acad. Sci. 494: 3 19-322. 2 1. SAINT-COME, C., G. R. ACKER, AND F. L. STRAND. 1982. Peptide influences on the development and regeneration of motor performance. Peptides 3: 439-449. 22. SAINT-COME, C., G. R. ACKER, AND F. L. STRAND. 1985. Development and regeneration of motor systemsunder the influence of ACTH peptides. Psychoneuroendocrinology 10: 445-459. 23. SAINT-COME, C., AND F. L. STRAND. 1985. ACTH 4-10 improves motor unit performance during peripheral nerve regeneration. Peptides 6(Suppl. 1): 77-83.
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24. SAINT-COME, C., AND F. L. STRAND. 1988. ACTH 4-9 analog (Org. 2766) improves qualitative and quantitative aspects of nerve regeneration. Peptides B(Supp1. 1): 215-22 1. 25. SKENE, J. H. P., R. D. JACOBSON,G. J. SNIPES,C. B. MCGUIRE, J. J. NORDEN, AND J. A. FREEMAN. 1986. A protein induced during nerve growth (GAP-43) is a major component of growth-cone membranes. Science 233: 783-785. 26. SETH, C. M., AND F. L. STRAND. 198 1. Neuromuscular response of the immature rat to ACTH/MSH 4-10. Peptides 2: 197-206. 27. STRAND, F. L., A. M. CAYER, E. GONZALEZ, AND H. STOBOY. 1976. Peptide enhancement of neuromuscular function: Animal and clinical studies. Pharmacol. Biochem. Behav. 5: 179-187. 28. STRAND, F. L., AND T. T. KUNG. 1980. ACTH accelerates recovery of neuromuscular function following crushing of peripheral nerve. Peptides 1: 135- 138. 29. STRAND, F. L., T. T. KUNG, AND C. SAINT-COME. 1981. Regenerative ability of spinal motor systems as influenced by ACTH/MSH peptides. Pages 369-409 in M. W. VAN HOF AND G. MOHN, Eds., Functional Recovery from Brain Damage. Elsevier/NorthHolland, Amsterdam. 30. STRAND, F. L., AND C. M. SMITH. 1986. LPH, ACTH, MSH and motor systems. Pages 245-27 1 in D. DE WIED, W. H. GISPEN, TJ. B. VAN WIMER~MA GREIDANUS, Eds., Neuropeptides and Behavior, Vol. 1. Pergamon Press, New York. 31. VERHAAGEN, J., P. M. EDWARDS, F. G. I. JENEKENS, P. SCHOTMAN, AND W. H. GISPEN. 1986. cr-Melanocyte-stimulating hormone stimulates the outgrowth of myelinated nerve fibers after peripheral nerve crush. Exp. Neurol. 92: 45 l-454. 32. VERHAAGEN, J., M. VAN HOOFF, P. EDWARDS, P. DE GRAAN, A. B. OE~TREICHER, P. SCHOTMAN, F. JENEKENS, AND W. GISPEN. 1986. The kinase C substrate protein B-50 and axonal regeneration. Brain Rex Bull. 17: 737-74 1. 33. ZWIERS, H., P. SCHOTMAN, AND W. H. GISPEN. 1980. Purification and some characteristics of an ACTH-sensitive protein kinasc and its substrate protein in rat brain membranes. J. Neurochem. 34: 1689-1699.
lw,531-541(1988)
ACTH Peptides Stimulate Motor Nerve Sprouting in Development RUTH E. FRISCHER AND FLEUR L. STRAND’ Center for Neuroscience, New York University, Washington Square, New York, New York 10003 Received September 8, I987 Maturational changes at the neuromuscular junction (nmj) of rat neonates were studied using scanning electron microscopy and light microscopy that permitted quantification of muscle fiber diameter, length of nerve terminal branching, end-plate area, and perimeter. Administration of ACTH 4- 10 ( 10 &kg S.C.daily from day of birth) stimulated nerve terminal branching, an effect most evident in 14&y-old pups. The trisubstituted derivative of ACTH 4-9 (Org 2766) when administered at 0.01 pg/ kg/daily, had a more potent effect, increasing end-plate perimeter and nerve terminal branching on the first postnatal week and markedly increasing only nerve terminal branching at 14 days of age. This is a dose-responsive action since 10 &kg/daily severely inhibits nerve sprouting. By 2 1 days, there were no differences between peptide- and saline-treated neonates. Peptide-induced sprouting was elicited only in the first 2 weeks of postnatal life. This time course corresponds with the critical period for nmj maturation and ceases when polyneuronal innervation of muscle fibers also terminates. It is suggested that ACTH peptides may exert a physiological role on nerve sprouting during development. 0 1988 Academic PI=. 1~.
INTRODUCTION Nerve sprouting occurs as a physiological process during normal development; it occurs as a restorative process following nerve crush or section. In the newborn rat each muscle fiber is polyneuronally innervated; i.e., each muscle fiber is contacted by several motor nerves. Typically during maturaAbbreviations: EDL-extensor digitorum longus, LM-light microscopy, nmj-neuromuscular junction, SEM-scanning electron microscopy. ’ This study was supported by The Council for Tobacco Research and by Grant BRSG RR07062 awarded by the Biomedical Research Support Grant Program of Research Resources, National Institutes of Health. We also thank Organon for ACTH 4-10 (ORG 0163) and ORG 2766. 531 0014-4886188 $3.00 Copyright 0 1988 by Academic Press, Inc. All rights of repmdwtion in any form reserved.
532
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STRAND
tion, the adult form of mono-neuronal innervation occurs between 2 and 3 weeks after birth (5, 18). In both intact adult nerve (28) and regenerating nerve (2,29,3 1) the presence of ACTH or ACTH peptide fragments stimulates the formation of nerve sprouts. While the causes of sprout formation during the process of regeneration appear to be better understood, the underlying mechanisms involved during development remain elusive. Both our electrophysiological studies and our studies on the development of motor behavior indicate that maturation of the neuromuscular system is accelerated by ACTH peptides ( 19,20,22). Using light and scanning electron microscopy (SEM) we have also shown that ACTH 4-10 and the ACTH 49 analog, Org 2766 (approximately 1000X more potent than ACTH 4-lo), have distinct neurotrophic effects, accelerating the morphological maturation of the developing rat neuromuscular junction (nmj) (10). In this study we asked whether this well-documented early maturation might be achieved through ACTH peptide effects on some phase of development such as axonal sprouting. We compared the effects of the two peptide fragments ACTH 4-10 and Org 2766, on structural parameters of the neuromuscular junction, including nerve terminal branching at the end-plate region, in neonatal rats 7, 14, and 2 1 days of age. METHODS
Animal Groups and Peptide Administration. Sprague-Dawley rats (dam plus pups) were maintained on a 12-h light/dark cycle and at a room temperature of 22°C. Each dam was housed separately and supplied with rat chow and water ad lib. On the day of birth (Day 1) each litter was culled to eight pups with approximately equal numbers of males and females. The pups were assigned randomly to litters and treated according to one of the following four protocols: 1. 0.9% saline 2. ACTH 4-10 (Org 0163), 10 pg/kg 3. Org 2766, the trisubstituted analog of ACTH 4-9,O.Ol &kg 4. Org 2766, 10 pg/kg was administered only in the series of experiments devoted to a SEM study of nerve terminal branching. As this dosage was found to be inhibitory, the lower dosage of 0.0 1 &kg, comparable to 10 &kg of ACTH 4- 10, was used for all other experiments. All injections were administered S.C. daily from Day 1. The animals were sacrificed at 7, 14, or 2 1 days of age. A series of untreated adult EDL muscles were processed for SEM for comparison purposes.
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Surgical Procedure. Pups were anesthetized with 40 mg/kg sodium pentabarbital i.p. before surgery. The animal was placed ventral side down on a dissecting table, a skin incision was made on the hind limb from the lateral epicondyle of the femur to the lateral malleolus in the ankle. The extensor digitorum longus (EDL) muscle was then exposed from the surrounding connective tissue. One EDL muscle was removed for the SEM studies, the contralateral muscle being used for light microscopy (LM). SpecimenPreparation for SEM. The muscle was fixed in situ and prepared for SEM using modifications of both Desaki and Uehara (8) and Fahim et al. (9). Endplates were localized with a cholinesterase stain, dissected free, postfixed in 2% unbuffered osmium tetroxide, and rinsed in distilled water. The extraneous cutaneous tissue was removed with 8 N HCl treatment in 60°C until the tissue separated into individual muscle fibers. The fibers were collected with a 5-ml glass syringe and allowed to flow onto an 0.8-pm poresize Nucleopore filter. The specimens were then washed with distilled water, dehydrated through a graded series of ethanol, critical-point dried, coated with carbon and gold palladium, and examined in an AMR 1000 scanning electron microscope at 20 kV. Specimen Preparation for LM. Intramuscular nerves and end plates were visualized by the combined silver-cholinesterase method of Pecot-Dechavassine et al. (17). The EDL muscle was fixed in situ by the application of 1% neutral formalin in normal Ringer’s solution. After excision, the EDL muscle was placed in fresh 1% neutral formalin for 3-4 min. The end plates were localized with a cholinesterase stain. The whole muscle was fixed in 5% neutral formalin, placed in 10% Triton X- 100, and postfixed in 80% ethanol. After impregnation with 0.5% Protargol the silver stain was reduced by the use of a modified Bodian developer. The whole muscle was rinsed in distilled water, finely teased, and mounted on slides. Quantitative Morphometric Analyses of LM Preparations. LM preparations were observed using an Olympus BH 100X oil immersion objective. A low-light-sensitive video camera mounted on top of the microscope projected the slide image onto a monitor. A Bioquant image digitizer with a manually operated cursor was used to determine measurements. Parameters measured included muscle fiber diameter, total length of nerve terminal branching, and cholinesterase-positive end-plate area and perimeter. Eight nmjs per neonate were measured. Each group consisted of five neonates for a total of 40 nmjs per group. An example of an outline graphics of digitized nmjs is demonstrated in Fig. 1. Data and outline graphics of all end-plate measurements were automatically fed into an Apple IIe computer. The Student t test was used for statistical evaluation. Data are expressed as means and standard error of the mean.
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FIG. 1. An example of a computer graphic outline of neuromuscular junctions after obtaining morphometric parameters. The numbers on the outline correspond to one of eight neuromuscular junctions measured.
RESULTS Scanning Electron Microscopy. During development there is a progressive change in the pattern of innervation of the muscle. The polyneuronal innervation of the immature skeletal muscle is shown in Fig. 2A. For comparison, the innervation of each muscle fiber by a single nerve, the characteristic pattern of the adult motor unit is shown in Fig. 2B. Figure 2C indicates the complexity of nerve terminal attachments at the adult nmj. When neonatal animals are treated with ACTH 4-10 (10 &kg) or Org 2766 (0.01 pg/kg) during the first 2 weeks of life, arborization of the nerve terminals entering the EDL is markedly increased (Fig. 3C-E) as compared to saline-treated controls (Fig. 3A). The inhibitory effect of the higher dosage of ORG 2766 (10 pg/kg) on nerve branching is clearly seen in Fig. 3B. Quantification of the three dimensional views of the developing end plates is difficult: peptide treatment appears to increase nerve terminal branching, a subjective interpretation that is borne out by the morphometric analysis of the LM preparations. Morphometric Analysis of LM Preparations. Table 1 shows that as the saline-treated neonate matures from 7 to 14 to 2 1 days of age, there are clear increases in muscle fiber diameter, end-plate area and perimeter, and nerve terminal branching. Treatment with ACTH 4- 10 ( 10 pg/kg) results in a diminution of muscle fiber diameter (P < 0.01) when measurements are made at 7 days of age. End-plate area and nerve terminal branching are similarly reduced (P < 0.05) at this time in development. No effect on end-plate perimeter is evident. However, Org 2766 at this stage in development increases
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FIG. 2. A-several nerves converging at a neuromuscular junction site on EDL muscle fiber of 7-day-old rat. 2440X. B-a motor unit of the peroneal nerve is seen branching and innervating several EDL muscle fibers. 220X. C-an adult rat end plate showing the complexity of nerve terminal attachment. 3050X.
both end-plate perimeter and nerve terminal branching. When peptide treatment is continued to Day 14, it is clear that both ACTH peptides stimulate nerve terminal branching, with Org 2766 being considerably more effective
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FIG. 3. Scanning electron micrographs of end-plate regions from the EDL muscles of 15 day-old rats. A-saline-treated neonate. The nerve can be seen branching into narrower nerve terminals along the muscle fiber. 188 1X. B-ORG 2766-treated (10 &kg) neonate. The nerve terminal shows a marked reduction in branching. 1986X. C-ACTH 4-lo-treated (10 pplkg) neonate showing extensive nerve terminal branching. 1100X. D-ORG 2766~treated (0.01 ag/ kg) neonate with arborization of nerve terminals. 2750X. E-higher magnification of ORG 2766-treated (0.01 &kg) neonate showing considerable nerve terminal branching at the end plate. 5775X.
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TABLE I Endplate Measurements from EDL Muscles of Rats Treated with ACTH/MSH 4- 10 (10 &kg), ORG 2766 (0.01 &kg), or Saline Daily S.C.from Day of Birth Age 7 Days Saline 4-10 2766 14 Days Saline 4-10 2766 2 1 Days Saline 4-10 2766
Muscle fiber diameter
End-plate area
End-plate perimeter
(rm)
(wd
(0)
Nerve terminal branching (gm)
32.1 +- 0.7 29.4 f 0.5** 32.9 f 0.9
1010.8 + 42.1 907.2 + 27.7* 1084.0 f 40.6
126.1 _t 2.1 122.3 k 2.2 137.7 f 2.5’*
108.3 -e 3.9 94.6 + 3.8+ 122.2 f 4.1*
38.2 + 0.9 38.2 + 0.9 38.5 f 0.9
1556.8 + 82.9 1631.9 f 63.6 1792.9 f 104.8
160.5 k 4.0 159.0 + 2.9 166.9 + 4.8
146.4* 6.7 172.4 + 8.0** 192.7 + 7.7***
55.1 * 1.2 53.9 f 1.4 52.3 + 1.1
2721.8 + 108.6 2686.0 k 101.8 2942.6 + 101.5
209.3 + 4.2 211.1 t4.1 220.4 + 4.2
296.6 + 8.3 273.6 i 10.1 294.6 f 8.7
Note. Values are means f SE. n = 40. * P < 0.05. **p
(P < 0.001) than ACTH 4-10 (P < 0.05) in this regard (Table 1). However, at this age there is little difference between the saline-treated controls and those animals administered either ACTH peptide when muscle fiber diameter, end-plate area, and perimeter are analyzed. By 2 1 days animals administered either ACTH peptide (when muscle fiber diameter, end-plate area, and perimeter are analyzed) do not differ from the saline controls in any of these morphological parameters. DISCUSSION These results confirm our earlier observations that ACTH 4- 10 is primarily neurotrophic in action (13,27). We show here that of all the parameters investigated, only nerve terminal branching at 14 days of age is effectively stimulated by this peptide. Muscle fiber diameter and end-plate area are actually reduced at 7 days of age and are subsequently unresponsive to ACTH 4- 10. The ACTH 4-9 analog, Org 2766, on the other hand, does not evoke these early presumably deleterious effects on muscle fiber diameter and endplate area, increasing both end-plate perimeter and nerve terminal branching at 7 days of age and stimulating nerve terminal branching still more effectively at 2 weeks of age. This action is dose-responsive, with 0.01 &kg being
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stimulatory and 10 pg/kg markedly inhibitory, a result often incurred with peptide administration. Electrophysiological studies clearly demonstrate that Org 2766 improves both the quality and rapidity of muscle reinnervation (24). During both development and regeneration, the presence of ACTH or ACTH peptide fragments stimulates the formation of nerve sprouts. In the developing neuromuscular system, this is seen as an increase in peripheral axonal arborization at the end-plate region (1). The present studies on the developing nmj describe an age-related increase in nerve terminal branching both prior to entering the immature muscle (Fig. 3) and within the end plate (Table l), correlating to the period of polyneuronal innervation. Therefore, the process of arborization is enhanced by peptide treatment during the first 2 weeks of postnatal life, with Org 2766 being a more potent stimulus for nerve branching than ACTH 4-10. Stimulatory effects of ACTH peptides on nerve outgrowth following nerve crush have been demonstrated by Gispen and his colleagues (3,7,3 1). These investigators also reported that of the melanocortin family of peptides, a-MSH (ACTH 1- 13) and its analogs have more potent neurotrophic properties than the corticotrophic moiety ACTH 4- 10 or its fragments. None of these peptides have any significant adrenocortical-stimulating activity ( 15). One of several possible modes of action to explain the acceleration of nerve sprouting induced by ACTH peptides in both development and regeneration involves a suggestion put forward by Gispen (12): namely, that following binding by the peptide to nerve-specific sites, calcium-dependent phosphorylation of specific synaptic plasma membrane proteins is inhibited. One of these proteins is B-50, which appears to be identical to GAP-43 and protein F 1 ( 16) phosphoproteins also associated with synaptic membranes. B-50 immunoreactivity is considerably increased in the regenerating peripheral nerves and localized in their newly formed sprouts and in the neuromuscular junctions (32). Of interest to any hypothesis that attempts to correlate mechanisms involved in regeneration and development is that maximal B-50 synthesis corresponds approximately to periods of profuse axonal outgrowth and synaptogenesis during ontogeny. During the development of the mammalian central nervous system, B-50 synthesis peaks early in the second postnatal week (14,25). It is also during these crucial first 2 weeks of postnatal life that the infant rat neuromuscular system is responsive to ACTH peptide administration ( 1, 10, 11,20-22,26). Synthesis of this growth-associated protein rapidly declines after this time, as does susceptibility to peptide action. The problem with this concept is that B-50 phosphorylation has not been shown to be sensitive to the smaller ACTH fragments such as ACTH 4- 10; sensitivity in the regenerating nerve appears to be limited to wMSH (ACTH l-l 3) and ACTH l-24 (33). The possibility that the developing nervous system may
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respond to the smaller ACTH fragments remains to be investigated. Other possible mechanisms that may explain our results include the suggestion that ACTH may evoke axonal sprouting at critical developmental periods by influencing neuronal responsiveness to some local target-tissuereleased trophic substance (4). ACTH may act in concert with a trophic substance and thus synergistically evoke nerve terminal sprouting by generating a permissive environment for sprouting to occur. ACTH can act only briefly in this capacity as there is a very small window in time that is open in the course of nmj development wherein sprouting can occur. It has been clearly shown that in regenerating systems, ACTH peptides effectively accelerate motor and sensory recovery only if peptide administration is begun within 48 hr after nerve crush (6,24). As administration immediately following the lesion is still better, it would appear that the melanocortins act on early growth processes (3 1) and that sensitivity to these peptides is indeed timedependent. In this morphological study all peptide-induced changes are evident only during the first 2 weeks of postnatal life: peptide acceleration of nmj maturation brings these parameters to a level at 2 weeks of age which is reached by the saline-treated controls at only 3 weeks of age. This demonstrates once again, as in our behavioral and electrophysiological studies (1, 19,20-22,27, 30), that neuropeptides bring a developing, regenerating, or depressed system to an optimal physiological level but do not elevate it to superlevels. SUMMARY In this paper we report that ACTH 4-10 and ORG 2766 stimulate nerve terminal sprouting most significantly at 14 days of age in the rat. The time course of this effect correlates with the maturational processes involved in neuromuscular development and ceases during the termination of polyneuronal innervation. This suggests that the stimulation for motoneuron terminal sprouting can be facilitated and further promoted by the exogenous administration of ACTH peptides at critical developmental periods and indicates a physiological role for these neurotrophic peptides in nerve sprouting. REFERENCES 1. ACKER, G. R., R. E. FRISCHER,AND F. L. STRAND. 1984. ACfH peptide neuromodulation in the developing neuromuscular system as seen through three different perspectives. Ann. N. Y. Acad. Sci. 435: 370-375. 2. BJJLSMA, W. A., F. G. I. JENEKENS, P. S~HOTMAN, AND W. H. GISPEN. 1983. Stimulation by ACTH 4- 10 of nerve fiber regeneration following sciatic nerve crush. Muscle Nerve 6: 104-l 12. 3. BIJLSMA, W. A., P. SCHOTMAN, F. G. I. JENEKENS,W. H. GISPEN, AND D. DE WIED. 1983. The enhanced recovery of sensorimotor function in rats is related to the melanotropic moiety of ACTH/MSH neuropeptides. Eur. J. Pharmacol. 9: 231-236.
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4. BROWN, M. C., R. L. HOLLAND, AND W. H. HOPKINS. 198 I. Motor nerve sprouting. Annu. Rev. Neurosci. 4: 11-42. 5. BROWN, M. C., J. K. S. JANSEN, AND D. VAN ESSEN. 1976. Polyneural innervation of skeletal muscle in new born rats and its elimination during maturation. J. Physiol. 261: 387-422. 6. DEKKER, A. J. A. M., M. M. PRINCEN,H. DE NIJS, L. G. J. DE LEEDE, AND C. L. E. BROEKKAMP. 1987. Acceleration of recovery from sciatic nerve damage by the ACIH (4-9) analog Org 2766: Different routes of administration. Peptides 8: 1057-1060. 7. DE KONING, P., J. H. BRAKEE, AND W. H. GISPEN. Methods for producing a reproducible crush in the sciatic nerve of the rat and the rapid and precise testing of return of sensory function: Beneficial effectsof melanocortins. J. Neurol. Sci. 14: 237-246. 8. DESAKI, J., AND Y. UEHARA. 198 1. The overall morphology of neuromuscular junctions as revealed by scanning electron microscopy. J. Neurocytol. 10: 10 1- 110. 9. FAHIM, M. A., J. A. HOLLEY, AND N. ROBBINS. 1983. Scanning and light microscopic study of age changes at a neuromuscular junction in the mouse. J. Neurocytol. 12: 12-25. 10. FRISCHER,R. E., N. EL-KAWA, AND F. L. STRAND. 1985. ACTH peptides as organizers of neuronal patterns in development: Maturation of the rat neuromuscular junction as seen by scanning electron microscopy. Peptides 6(Suppl. 2): 13- 19. 11. FRISCHER,R. E., AND F. L. STRAND. 1988. Neural effects of ACTH peptide treatment on the developing rat neuromuscular junction. Ann. N. Y. Acad. Sci., in press. 12. GISPEN, W. H., C. J. VAN DONGEN, P. N. E. DEGRAAN, A. B. OFSTREICHER, AND H. ZWIERS. 1985. The role of phosphoprotein B-50 in phosphoinositide metabolism in brain synaptic plasma membranes. Pages 399-413 in J. E. BLEASDALE, G. HAWSER, AND J. EICHBERG,Eds., Inositol and Phosphoinositides. Humana, Clifton, NJ. 13. GONZALEZ, E. R., AND F. L. STRAND. 198 1. Neurotropic action of ACTH/MSH 4- 10 on neuromuscular function in hypophysectomized rats. Peptides 2(Suppl. 1): 107- 113. 14. JACOBSON,R. D., I. VIRAG, AND J. H. P. SKENE. 1986. A protein associated with axon growth, GAP-43, is widely distributed and developmentally regulated in rat CNS. J. Neurosci. 5: 1843-1855. 15. LANDE, S., D. DE WIED, AND A. WITHER. 1973. Unique pituitary peptides with behavioraffecting activity. Prog. Brain Res. 39: 42 l-427. 16. NELSON, R. B., AND A. ROUTTENBERG. 1985. Characterization of protein Fl(47 kDa, 4.5 pl): A kinase C substrate directly related to neural plasticity. Exp. Neurol. 89: 2 13-224. 17. PECOT-DECHAVASSINE, M., A. WERNIG, AND H. STOVER. 1979. A combined silver and cholinesterase method for studying exact relations between the pm- and the postsynaptic elements at the frog neuromuscular junction. Stain Technol. 54: 25-27. 18. REDFERN, P. A. 1970. Neuromuscular transmission in newborn rats. J. Physiol. 209: 701709. 19. ROSE, K. J., R. E. FRISCHER,J. A. KING, AND F. L. STRAND. Neonatal neuromuscular parameters vary in susceptibility to postnatal ACTH 4-10 administration. Peptides, in press. 20. ROSE, K. J. AND F. L. STRAND. 1987. Response of the developing neuromuscular system of the rat to nicotine and the neurotropic peptide fragment ACTH/MSH 4-10. Ann. N. Y. Acad. Sci. 494: 3 19-322. 2 1. SAINT-COME, C., G. R. ACKER, AND F. L. STRAND. 1982. Peptide influences on the development and regeneration of motor performance. Peptides 3: 439-449. 22. SAINT-COME, C., G. R. ACKER, AND F. L. STRAND. 1985. Development and regeneration of motor systemsunder the influence of ACTH peptides. Psychoneuroendocrinology 10: 445-459. 23. SAINT-COME, C., AND F. L. STRAND. 1985. ACTH 4-10 improves motor unit performance during peripheral nerve regeneration. Peptides 6(Suppl. 1): 77-83.
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24. SAINT-COME, C., AND F. L. STRAND. 1988. ACTH 4-9 analog (Org. 2766) improves qualitative and quantitative aspects of nerve regeneration. Peptides B(Supp1. 1): 215-22 1. 25. SKENE, J. H. P., R. D. JACOBSON,G. J. SNIPES,C. B. MCGUIRE, J. J. NORDEN, AND J. A. FREEMAN. 1986. A protein induced during nerve growth (GAP-43) is a major component of growth-cone membranes. Science 233: 783-785. 26. SETH, C. M., AND F. L. STRAND. 198 1. Neuromuscular response of the immature rat to ACTH/MSH 4-10. Peptides 2: 197-206. 27. STRAND, F. L., A. M. CAYER, E. GONZALEZ, AND H. STOBOY. 1976. Peptide enhancement of neuromuscular function: Animal and clinical studies. Pharmacol. Biochem. Behav. 5: 179-187. 28. STRAND, F. L., AND T. T. KUNG. 1980. ACTH accelerates recovery of neuromuscular function following crushing of peripheral nerve. Peptides 1: 135- 138. 29. STRAND, F. L., T. T. KUNG, AND C. SAINT-COME. 1981. Regenerative ability of spinal motor systems as influenced by ACTH/MSH peptides. Pages 369-409 in M. W. VAN HOF AND G. MOHN, Eds., Functional Recovery from Brain Damage. Elsevier/NorthHolland, Amsterdam. 30. STRAND, F. L., AND C. M. SMITH. 1986. LPH, ACTH, MSH and motor systems. Pages 245-27 1 in D. DE WIED, W. H. GISPEN, TJ. B. VAN WIMER~MA GREIDANUS, Eds., Neuropeptides and Behavior, Vol. 1. Pergamon Press, New York. 31. VERHAAGEN, J., P. M. EDWARDS, F. G. I. JENEKENS, P. SCHOTMAN, AND W. H. GISPEN. 1986. cr-Melanocyte-stimulating hormone stimulates the outgrowth of myelinated nerve fibers after peripheral nerve crush. Exp. Neurol. 92: 45 l-454. 32. VERHAAGEN, J., M. VAN HOOFF, P. EDWARDS, P. DE GRAAN, A. B. OE~TREICHER, P. SCHOTMAN, F. JENEKENS, AND W. GISPEN. 1986. The kinase C substrate protein B-50 and axonal regeneration. Brain Rex Bull. 17: 737-74 1. 33. ZWIERS, H., P. SCHOTMAN, AND W. H. GISPEN. 1980. Purification and some characteristics of an ACTH-sensitive protein kinasc and its substrate protein in rat brain membranes. J. Neurochem. 34: 1689-1699.